Over the last project period, we have achieved the goal of detecting the expression of many genes in a single cell using multiplexed probes to develop a barcode for eleven transcription sites for specific genes and their alleles. This single cell gene expression profiling method allows assessment of each cell's pattern of expression as well as allowing a population analysis of the regulation of these genes. The extensive data complexity that results from the gene-to-gene analyses indicates that this will be an informative approach to validate the gene expression patterns pertaining to cancer gene expression as well as to discover new correlations of genes with the cancer phenotype.
The aim of this proposal is to apply this methodology we have developed to tissue samples in order to derive specific gene expression information, ultimately from patient specimens. The approach is to optimize this technology by developing reagents, protocols and imaging hardware and software to achieve a high signal of expressed genes and reduced background of tissue autofluorescence. Central to this development is the use of two new instruments, the Leica confocal microscope, and the VarispecTM liquid crystal tunable filter, which allow a complete spectral analysis of the hybridized tissue, and hence extraction of the principal dye components used for the probe. This provides a means by which we can multiplex the barcoding probes using more spectral bandwidth and obtain gene expression profiling of up to 247 genes in a single nucleus. The statistical analysis of the expression of these genes in tissue combined with a preserved tissue morphology will provide an eventual platform for interrogation of patient specimens where the outcomes are known. The expression patterns obtained from tumor cells in pathological samples will allow a correlation of specific genes with disease progression.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Exploratory/Developmental Grants Phase II (R33)
Project #
5R33CA083208-06
Application #
6946512
Study Section
Special Emphasis Panel (ZCA1-SRRB-C (J1))
Program Officer
Rasooly, Avraham
Project Start
1999-08-11
Project End
2007-06-30
Budget Start
2005-09-15
Budget End
2006-06-30
Support Year
6
Fiscal Year
2005
Total Cost
$549,338
Indirect Cost
Name
Albert Einstein College of Medicine
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
110521739
City
Bronx
State
NY
Country
United States
Zip Code
10461
Wang, Donghai; Pezo, Rossanna C; Corner, Georgia et al. (2010) Altered dynamics of intestinal cell maturation in Apc1638N/+ mice. Cancer Res 70:5348-57
Gu, Wei; Pan, Feng; Singer, Robert H (2009) Blocking beta-catenin binding to the ZBP1 promoter represses ZBP1 expression, leading to increased proliferation and migration of metastatic breast-cancer cells. J Cell Sci 122:1895-905
Maier, Sandra; Daroqui, M Cecilia; Scherer, Stefan et al. (2009) Butyrate and vitamin D3 induce transcriptional attenuation at the cyclin D1 locus in colonic carcinoma cells. J Cell Physiol 218:638-42
Pezo, Rossanna C; Gandhi, Saumil J; Shirley, L Andrew et al. (2008) Single-cell transcription site activation predicts chemotherapy response in human colorectal tumors. Cancer Res 68:4977-82
Capodieci, Paola; Donovan, Michael; Buchinsky, Heidi et al. (2005) Gene expression profiling in single cells within tissue. Nat Methods 2:663-5
Levsky, Jeffrey M; Shenoy, Shailesh M; Pezo, Rossanna C et al. (2002) Single-cell gene expression profiling. Science 297:836-40
Wilson, Andrew J; Velcich, Anna; Arango, Diego et al. (2002) Novel detection and differential utilization of a c-myc transcriptional block in colon cancer chemoprevention. Cancer Res 62:6006-10